GH is a member of a large family of hormones involved in the regulation of mammalian growth and development. Human GH is a 22 kDa polypeptide which is involved in a number of biological processes. For example, cell growth, lactation, the activation of macrophages and the regulation of energy metabolism. GH interacts sequentially with two membrane bound GHR's via two separate sites on GH referred as site 1 and site 2. Site 1 is a high affinity binding site and site 2 a low affinity site. A single GH molecule binds 1 GHR via site 1. A second GHR is then recruited via site 2 to form a GHR:GH:GHR complex. The complex is then internalised and activates a signal transduction cascade leading to changes in gene expression.
The extracellular domain of the GHR exists as two linked domains each of approximately 100 amino acids (SD-100), the C-terminal SD-100 domain (b) being closest to the cell surface and the N-terminal SD-100 domain (a) being furthest away. It is a conformational change in these two domains that occurs on hormone binding with the formation of the trimeric complex GHR-GH-GHR.
Modified GH's are disclosed in U.S. Pat. No. 5,849,535 which is incorporated by reference. The modification to GH is at both site 1 and site 2 binding sites. The modifications to site 1 produce a GH molecule which has a higher affinity for GHR compared to wild-type GH. These modified GH molecules act agonists. There is also disclosure of site 2 modifications which result in the creation of GH antagonists. Further examples of modifications to GH which alter the binding affinity of GH for site 1 are disclosed in U.S. Pat. Nos. 5,854,026; 6,004,931; 6,022,711; 6,057,292; and 6,136,563 each of which are incorporated by reference. A summary of the modifications made to site 1 is provided in Table 1. Modifications to site 2 are also disclosed, in particular amino acid residue G120 which when modified to either arginine, lysine, tryptophan, tyrosine, phenylalanine, or glutamic acid creates a GH molecule with antagonistic properties.
In addition, the modified GH is coated in polyethylene glycol (PEG) by a process known as “pegylation” this has several beneficial effects. Firstly, the PEG coat increases the effective molecular weight of GH from 22 kD to approximately 40 kD. The effect this has is to decrease glomerular filtration of GH thereby increasing the half-life of GH in vivo which reduces the dose administered to produce the desired effect. In addition pegylation is thought to reduce both the immunogenicity and toxicity of proteins which are treated in this way, see Abuchowski et al J Biol Chem., 252, 3578-3581, (1977).
However, a consequence of pegylation is to reduce the affinity of the modified GH molecule for GHR. This means that an increased dose is required to counter the reduced affinity. This is undesirable since it counteracts the advantageous effect of pegylation with respect to increasing the half life of modified GH. It would be desirable to provide a modified GH molecule which does not require pegylation but has an increased half-life and also has the added benefits of reduced immunogenicity and lacks toxicity.